Journal: Applied Microbiology and Biotechnology

Article Title: Enhancing production and assessing IgE reactivity of dog allergen Can f 6 in Pichia pastoris and Escherichia coli

doi: 10.1007/s00253-025-13465-7

Figure Lengend Snippet: E. coli -produced recombinant Can f 6 variant expression and purification. a, b Schematic illustrations of gene expression constructs. a pET28a( +)-Can f 6 expression vector features the Can f 6 gene tagged at the N-terminus with a 6xHis tag. b pET28a( +)-MBP-TEV-Can f 6 expression vector features Can f 6 fused to the C-terminus of a 6xHis-MBP fusion protein, with a TEV protease cleavage site inserted between the fusion partner and the target protein. SDS-PAGE analysis of expression and purification of c rCan f 6 and d rMBP-Can f 6. Lanes: (1), lysate fraction from non-induced E. coli ; (2), lysate fraction from IPTG-induced E. coli ; (3), soluble fraction before purification with nickel-chelate chromatography; (4), soluble fraction after incubation with Ni–NTA agarose; (5–9), wash fractions; (10–14), elution fractions containing purified rCan f 6 and rMBP-Can f 6 proteins, respectively. M—Pierce™ Unstained Protein MW Marker (Thermo Fisher Scientific, USA). Proteins were separated on SDS-PAGE gels of c 12% and d 14% acrylamide concentrations

Article Snippet: Subsequent cloning of the resultant DNA fragment occurred at the BamH I and Xho I sites within pET28a( +) and pET28a( +)-MBP-TEV vectors, gifts from Zita Balklava & Thomas Wassmer (Addgene plasmid #69,929; http://n2t.net/addgene:69929 ; RRID:Addgene_69929) (Currinn et al. ).

Techniques: Produced, Recombinant, Variant Assay, Expressing, Purification, Gene Expression, Construct, Plasmid Preparation, SDS Page, Chromatography, Incubation, Marker

Journal: Animals : an Open Access Journal from MDPI

Article Title: Recombinant Polymerase Amplification Coupled with CRISPR/Cas12a Detection System for Rapid Visual Detection of Porcine Circovirus 3

doi: 10.3390/ani14172527

Figure Lengend Snippet: The development of the two-pot RPA-CRISPR/Cas12a-PCV3 detection method. ( A , B ) The measurement of the CRISPR/Cas12a reaction targeting PCV3 DNA at various times (5, 10, 20, 30 or 40 min) by blue light, UV light ( A ), and fluorescence signal ( B ). ( C ) Schematic diagram of two-pot RPA-CRISPR/Cas12a-PCV3 detection method. The data are expressed from three independent experiments.

Article Snippet: A full-length fragment of Cas12a was amplified from the 6 His-MBP-TEV-huLbCas12a plasmid (#90096; Addgene, Beijing, China) and cloned into the pET-28a vector to generate the pET-28a-Cas12a expression plasmid.

Techniques: CRISPR, Fluorescence

Journal: Animals : an Open Access Journal from MDPI

Article Title: Recombinant Polymerase Amplification Coupled with CRISPR/Cas12a Detection System for Rapid Visual Detection of Porcine Circovirus 3

doi: 10.3390/ani14172527

Figure Lengend Snippet: The sensitivity of two-pot RPA-CRISPR/Cas12a-PCV3 detection method. ( A , B ) The sensitivity of two-pot method detection with 10-fold serially diluted viral DNA (concentrations 2 × 10 11 , 2 × 10 9 , 2 × 10 7 , 2 × 10 5 , 2 × 10 3 , 2 × 10 1 , and 2 × 10 0 copies/µL, respectively) by blue light, UV light ( A ), and fluorescence signal ( B ). The data are expressed as the means ± SDs from three independent experiments (*** p < 0.001).

Article Snippet: A full-length fragment of Cas12a was amplified from the 6 His-MBP-TEV-huLbCas12a plasmid (#90096; Addgene, Beijing, China) and cloned into the pET-28a vector to generate the pET-28a-Cas12a expression plasmid.

Techniques: CRISPR, Fluorescence

Journal: Animals : an Open Access Journal from MDPI

Article Title: Recombinant Polymerase Amplification Coupled with CRISPR/Cas12a Detection System for Rapid Visual Detection of Porcine Circovirus 3

doi: 10.3390/ani14172527

Figure Lengend Snippet: The specificity of two-pot RPA-CRISPR/Cas12a-PCV3 detection method. ( A ) The specific PCR products of some swine viruses (PCV3, PCV1, PCV2, PCV4, PRRSV, PRV, SVA, PEDV, and CSFV). ( B , C ) The specificity of two-pot method detection with four genotypes of PCV (PCV3, PCV1, PCV2, and PCV4) by blue light, UV light ( B ), and fluorescence signal ( C ). ( D , E ) The specificity of two-pot method detection with some swine viruses (PCV3, PRRSV, PRV, SVA, PEDV, and CSFV) by blue light, UV light ( D ), and fluorescence signal ( E ). NTC represents no-viral DNA or cDNA control. The data are expressed as the means ± SDs from three independent experiments (**** p < 0.0001).

Article Snippet: A full-length fragment of Cas12a was amplified from the 6 His-MBP-TEV-huLbCas12a plasmid (#90096; Addgene, Beijing, China) and cloned into the pET-28a vector to generate the pET-28a-Cas12a expression plasmid.

Techniques: CRISPR, Fluorescence, Control

Journal: Animals : an Open Access Journal from MDPI

Article Title: Recombinant Polymerase Amplification Coupled with CRISPR/Cas12a Detection System for Rapid Visual Detection of Porcine Circovirus 3

doi: 10.3390/ani14172527

Figure Lengend Snippet: Comparison between two-pot RPA-CRISPR/Cas12a and qPCR detections for clinical samples.

Article Snippet: A full-length fragment of Cas12a was amplified from the 6 His-MBP-TEV-huLbCas12a plasmid (#90096; Addgene, Beijing, China) and cloned into the pET-28a vector to generate the pET-28a-Cas12a expression plasmid.

Techniques: Comparison

Journal: Animals : an Open Access Journal from MDPI

Article Title: Recombinant Polymerase Amplification Coupled with CRISPR/Cas12a Detection System for Rapid Visual Detection of Porcine Circovirus 3

doi: 10.3390/ani14172527

Figure Lengend Snippet: Detection of PCV3 clinical samples by two-pot RPA-CRISPR/Cas12a-PCV3 detection method. ( A ) The 25 clinical samples detection of two-pot RPA-CRISPR/Cas12a-PCV3 detection method by blue light, and UV light. ( B ) Comparison of detection results using two-pot RPA-CRISPR/Cas12a and qPCR-based PCV3 detection methods, with blue indicating negative results and orange indicating positive results. NTC represents no-viral DNA control.

Article Snippet: A full-length fragment of Cas12a was amplified from the 6 His-MBP-TEV-huLbCas12a plasmid (#90096; Addgene, Beijing, China) and cloned into the pET-28a vector to generate the pET-28a-Cas12a expression plasmid.

Techniques: CRISPR, Comparison, Control

Journal: Animals : an Open Access Journal from MDPI

Article Title: Recombinant Polymerase Amplification Coupled with CRISPR/Cas12a Detection System for Rapid Visual Detection of Porcine Circovirus 3

doi: 10.3390/ani14172527

Figure Lengend Snippet: The development of one-pot RPA-CRISPR/Cas12a-PCV3 detection method and its sensitivity. ( A ) Schematic diagram of one-pot RPA-CRISPR/Cas12a-PCV3 detection method. ( B , C ) The sensitivity of one-pot method detection with 10-fold serially diluted viral DNA (concentrations 2 × 10 11 , 2 × 10 9 , 2 × 10 7 , 2 × 10 5 , 2 × 10 3 , 2 × 10 1 , and 2 × 10 0 copies/µL, respectively) by blue light, UV light ( B ), and fluorescence signal ( C ). ( D , E ) The sensitivity of one-pot method detection with 2-fold serially diluted viral DNA (concentrations 2 × 10 1 , 1 × 10 1 , 0.5 × 10 1 , and 0.25 × 10 1 copies/µL, respectively) by blue light, UV ligh ( D ), and fluorescence signal ( E ). The data are expressed as the means ± SDs from three independent experiments (**** p < 0.0001).

Article Snippet: A full-length fragment of Cas12a was amplified from the 6 His-MBP-TEV-huLbCas12a plasmid (#90096; Addgene, Beijing, China) and cloned into the pET-28a vector to generate the pET-28a-Cas12a expression plasmid.

Techniques: CRISPR, Fluorescence

Journal: Animals : an Open Access Journal from MDPI

Article Title: Recombinant Polymerase Amplification Coupled with CRISPR/Cas12a Detection System for Rapid Visual Detection of Porcine Circovirus 3

doi: 10.3390/ani14172527

Figure Lengend Snippet: Detection of PCV3 clinical samples by one-pot RPA-CRISPR/Cas12a-PCV3 detection method. ( A ) The 25 clinical samples detection of one-pot RPA-CRISPR/Cas12a-PCV3 detection method by blue light and UV light. ( B ) Comparison of detection results between one- and two-pot RPA-CRISPR/Cas12a detection methods, with blue indicating negative results and orange indicating positive results. NTC represents no-viral DNA control.

Article Snippet: A full-length fragment of Cas12a was amplified from the 6 His-MBP-TEV-huLbCas12a plasmid (#90096; Addgene, Beijing, China) and cloned into the pET-28a vector to generate the pET-28a-Cas12a expression plasmid.

Techniques: CRISPR, Comparison, Control